Abstract

Dendritic cells (DCs), monocytes, and/or macrophages initiate host-protective immune responses to intracellular pathogens in part through interleukin-12 (IL-12) production, although the relative contribution of tissue resident versus recruited cells has been unclear. Here, we showed that after intraperitoneal infection with Toxoplasma gondii cysts, resident mononuclear phagocytes are replaced by circulating monocytes that differentiate in situ into inflammatory DCs (moDCs) and F4/80(+) macrophages. Importantly, NK cell-derived interferon-γ (IFN-γ) was required for both the loss of resident mononuclear phagocytes and the local differentiation of monocytes into macrophages and moDCs. This newly generated moDC population and not the resident DCs (or macrophages) served as the major source of IL-12 at the site of infection. Thus, NK cell-derived IFN-γ is important in both regulating inflammatory cell dynamics and in driving the local differentiation of monocytes into the cells required for initiating the immune response to an important intracellular pathogen.

Characterization of changes in peritoneal exudate cells (PEC) following infection

(A) Yet40 mice were left untreated (top) or challenged with T. gondii cysts (bottom) and PEC harvested 72 h post-infection followed by flow cytometry analysis. Plots shown are gated on total live PEC and are representative from individual animals. Gates #1 – 6 represent the gates utilized to sort the different PEC populations. Gates #1 – 2 were sorted from naïve animals and gates #3 – 6 were purified from infected mice. Gate #6 was determined using C57BL/6 infected mice as control for cell autofluorescence. See also .(B) As in A but histograms shown are gated on CD11c+MHC-II+ cells obtained from naïve (filled histogram) or infected (black line) mice. (A–B) Results shown are representative of three or more experiments performed (n= 3 – 6/group/experiment).(C) Giemsa staining of cytospin preparations from flow-cytometry-sorted populations described in A (600x original magnification). Dashed lines indicate different fields from the same cytospin preparation. Results shown are representative of three experiments performed; cells were pooled from 3 – 5 animals/group for purification.(D) Mixed leukocyte reaction of responder CD4+ T cells from BALB/c animals incubated with purified stimulating populations from C57BL/6 yet40 mice described in A at different S:R cell ratio. S: stimulator; R: responder. T cell proliferation was measured by [3H]-thymidine incorporation at 90h. The values shown are mean ± SD of triplicate cultures. One representative experiment of two performed is shown.(E) As in A but PEC were harvested at different time points after infection. Left panel shows absolute cell number of total PEC and bar graphs on the right show the absolute number of each cell population. Values shown are mean ± SD from a two or more experiments combined (n= 3 – 7/group/experiment).

Non-infected CD11b+CD8α− DCs represent the majority of IL-12p40 producing cells at the site of infection

(A) PEC from yet40 mice were harvested 72 h after infection and analyzed by flow cytometry. Left panels show the gating strategy on total YFP+ cells and dot plots on the right are gated on total YFP+ cells. Results shown are representative of three or more experiments performed (n= 3 – 6/group/experiment).(B) Frequency of peritoneal CD11c+MHCII+IL-12p40YFP+ at different time points post-infection. Values shown are mean ± SD from a minimum of three different experiments combined. *** p < 0.001.(C) As in A but dot plots show the frequency of IL-12p40YFP+ among CD11c+MHC-II+CD11b+ (left), CD11c+MHC-II+CD8α+ (middle) and CD11c+MHC-II+CD103+ (right) cells. Numbers indicate the frequency of each population within the gate. Dot plots are representative of 3 – 4 mice per group from 3 or more experiments performed.(D) Frequency of IL-12p40YFP+ cells among DCs subsets. Values shown are mean ± SD from 3 – 5 different experiments combined.(E) As in C but CD8α+ DCs were depleted by i.v. injection with anti-CD8α depleting antibody on days −1 and +1 relative to infection. Dot plots shown are from one representative experiment of 3 performed with 3 mice/group each. See also .

NK cell-derived IFNγ shapes the inflammatory infiltrate and is required for IL-12 production by peritoneal CD11b+CD8α− DCs

(A) Gating strategy for PEC sorting. PEC were obtained from anti-IFN-γ-treated mice 72 h after infection and three-way sorting was performed. Gates #7 – 9 represent the gates utilized for the sort. Cells from 3 – 4 mice were pooled for sorting. See also .(B) The morphology of purified populations obtained as described in A was evaluated on cytospin preparations following Giemsa staining (600x original magnification). Arrowheads show T. gondii parasites inside the cells. Dashed lines indicate different fields from the same cytospin preparation. One representative experiment of three performed is shown.(C) As in 1D but purified cells described in A were utilized as stimulating populations The values shown are mean ± SD of triplicate cultures of one representative experiment of two performed.(D) Surface expression of Ly6C (left) and CD11c (right) on purified CD11c+MHCII+ cells from naïve (filled histogram), anti-IFNγ-treated (black line) or untreated (blue line) infected mice and YFP+ cells (red line) from infected animals. Histograms are representative of one experiment out of three performed.(E) PEC were harvested 72 h post-T. gondii infection from WT yet40, WT yet40 that had received anti-IFN-γ a day before parasite challenge or Ifngr1−/− yet40 mice. Dot plots gated on CD11c+MHC-II+ cells, depict the frequency of IL-12p40YFP+ cells among CD11b+ (top) or CD8α+ (bottom) DCs subsets and are representative of individual animals from 3 – 4 mice per group and three or more experiments performed. Numbers indicate the frequency of each population in the corresponding quadrant.(F) Frequency of CD11c+MHCII+IL-12p40YFP+ in PEC from yet40 naïve or infected WT (Inf), infected + anti-IFN-γ (Inf + aIFNγ) or infected Ifngr1−/− yet40 mice. No differences were observed between naïve WT yet40 and naïve Ifngr1−/− yet40 animals. Values shown are mean ± SD from a minimum of three different experiments combined (n= 3 – 4/group/experiment). *** p < 0.001.(G) IL-12p40 production measured by ELISA in peritoneal exudate from naïve mice or 72 h post-infection of WT (Inf), WT treated with anti-IFN-γ (Inf + aIFNγ) or Ifngr1−/− animals. Values shown are mean ± SD of ELISA readings from 3 mice per group and are representative of three experiments performed. * p < 0.05.(H) IL-12p70 was measured by ELISA in peritoneal exudate from naïve animals, infected, infected treated with anti-IFN-γ (Inf + aIFNγ) or infected mice treated with anti-IFNγ and injected i.p with purified NK cells (aIFNγ + NK). Values shown are mean ± SD of ELISA readings from 3 mice per group and are representative of two (aIFNγ + NK) or three experiments performed. * p < 0.05.(I) PEC obtained 72 h after infection from yet40 mice (Infected; top), yet40 animals that were treated with anti-AsialoGM1 the day prior to infection (Infected + aAsialoGM1; middle) or ant-IFN-γ-treated yet40 mice that received purified NK cells (aIFNγ + NK; bottom) were analyzed by flow cytometry. Dot plots on the left are gated on total live PEC. Depletion of NK cells in Anti-AsialoGM1 treated mice is evidenced by the absence of DX5+/CD3− cells (middle panel). Dot plots on the right are gated on CD11c+MHC-II+ cells and show the frequency of IL-1240YFP+ cells. Dot plots shown are from individual animals from 3 – 4 mice per group of a representative experiment from two (aIFNγ + NK) or three experiments performed. Numbers indicate the frequency of cells in each quadrant.(J) Frequency of CD11c+MHCII+IL-12p40YFP+ in PEC from yet40 naïve, infected (Inf), Inf + anti-AsialoGM1 or aIFNγ + NK. Values shown are mean ± SD from a minimum two different experiments combined (n= 3 – 4/group/experiment). ** p < 0.01.

Transferred peritoneal macrophages and DCs fail to produce IL-12p40 and decrease in number following T. gondii infection

(A) Total PEC were obtained from naïve CD45.2+ yet40 animals and injected i.p. into CD45.1+ hosts naïve (left) or challenged with T. gondii 24 h earlier (right). PEC were harvested 72 h post-cell transfer. Donor-derived cells are indicated as CD45.2+. Dot plots are gated on total live PEC, frequency and absolute number (in parenthesis) of grafted CD45.2+ cells are indicated. No CD45.2+ cells were detected in CD45.1+ mice that did not receive yet40 PEC (not shown). See also .(B) Same as A but dot plots shown are gated on donor-derived CD45.2+ cells. Numbers indicate the frequency of cells within each gate. (A–B) dot plots shown are from representative individual animals of three experiments performed (n = 4 – 8).(C) Naïve yet40 PEC were transferred i.p. into CD45.1+ recipients a day after T. gondii infection and PEC were harvested at 24 h (left), 48 h (middle) or 72 h (right) following adoptive cell transfer. Dot plots shown are gated on CD45.2+ graft-derived cells and are representative of individual animals from 3 mice per group. Numbers indicate frequency of cells in each gate. No IL-12p40YFP+ cells were detected at any given time point (not shown).(D) PEC were harvested from Ccr2−/− naïve (top) or 72 h infected (bottom) animals. Dot plots shown are gated on total PEC after exclusion of dead cells, and represent individual animals from 3 mice/group/experiment from two experiments performed. Frequency and absolute number (in parenthesis) of cells within each gate are indicated.

(A) Purified BM monocytes were adoptively transferred i.v. into CD45.1+ hosts that were untreated (Naïve), infected a day earlier (Infected) or had been treated with anti-IFN-γ prior to T. gondii infection (Infected + aIFNγ). PEC were harvested 72 h after monocyte engraftment. Donor-derived cells are identified as CD45.2+CD45.1− among total live cells. An infected CD45.1+ host that did not receive yet40 monocytes is also shown as control for CD45.2+ specific gating. Dot pots shown are from individual animals from 4 – 5 mice per group of a representative experiment from two to six performed.(B) Same as A but graph shows the absolute number of CD45.2+ donor cells. Values shown are individual animals and the mean ± SD from two or three experiments combined with 4 – 5 mice/group/experiment are shown. * p < 0.05; *** p < 0.001.(C) As in A but graph represents the frequency of IL-12p40YFP+ cells among the donor monocyte-derived population. Values shown are individual animals with the mean ± SD from two or three experiments combined with 4 – 5 mice/group/experiment are indicated. ** p < 0.01; *** p < 0.001.(D) Similar to A but graph represents the mean fluorescence intensity (MFI) of F4/80 expression on donor-derived cells. Values shown are individual animals with the mean ± SD from two or three experiments combined with 4 – 5 mice/group/experiment are indicated. *** p < 0.001.(E) Phenotypic analysis of CD45.2+ monocyte-derived cells. PEC harvested from mice as described in A but dot plots are gated on CD45.2+ grafted cells. Dot plots shown are from individual animals from 4 –5 mice/group/experiment of a representative experiment from two or more performed. Numbers indicate frequency of cells within each quadrant.

(A–D) PEC were obtained from naïve yet40 mice, infected (Inf.) or infected that had been treated with anti-IFN-γ prior parasite challenge (Inf. + aIFNγ) followed two, three or four-way sorting as described in and . BM monocytes from naïve animals were purified as described in . Purified populations obtained from three independent experiments were analyzed by Nanostring technology. (A, C–D) Average-linkage–based non-supervised hierarchical clustering of most significant genes (q<0.001) differentially expressed between all samples (A; n=104), inflammatory monocytes/macrophages (C; n=46) and dendritic cells (D; n=45). Each gene expression was normalized to its mean of expression of the samples in the analysis and standard deviation was scaled to the value of one. Red indicates expression above, green below and black-no difference from the mean of all samples.(B) Data were also visualized using unsupervised Principal Component Analysis. Normalized expression data of all genes (n=127; see Experimental Procedures) and all samples used without prior selection of the genes. See also .